Taking advantage of recent developments in the field of metallic nanoparticle-based colorimetric DNA detection and in the field of in vitro selection of functional DNA/RNA that can recognize a wide range of analytes, we have designed highly sensitive and selective colorimetric biosensors for many analytes of choice. As an example of the sensor design strategy, a highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reviewed. The DNAzyme consists of an enzyme and a substrate strand, which can be used to assemble DNA-functionalized gold nanoparticles. The aggregation brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage of the substrate strand, which disrupts the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The application of the sensor in lead detection in leaded paint is also demonstrated. In perspective, the use of allosteric DNA/RNAzymes to expand the range of the nanoparticle-based sensor design method is described.